Anemonefish are a group of 28 species of coral reef fish belonging to the family Pomacentridae, subfamily Amphiprioninae, all characterized by living in symbiosis with sea anemones of several genera. Some anemonefish are specialized to cooperate with a single or few species of sea anemone, being immune to their poisonous tentacles but sensible to those of other species of sea anemones, while other anemonefish are more generalist and able to live together with a number of different species of sea anemone hosts. Despite the common life style, anemonefish species occur in a variety of colors, body shapes and degree of dependence from the host. To understand the evolutionary mechanisms responsible for the anemonefish diversification, we studied 23 out of 28 species of anemonefish by analyzing three mitochondrial regions: the cytochrome b gene, the 16S ribosomal RNA gene and the first half of the D-loop, a non-coding, regulatory region to reconstruct their molecular phylogeny through Bayesian and maximum parsimony approaches. The evolution of specialization was studied by means of character reconstruction methods. This work includes the highest number of anemonefish so far analyzed and particularly some species that had never been studied before. The results support a monophyletic origin for the subfamily Amphiprioninae, in contrast to the current taxonomy, based on morphological characters, that divides anemonefish into two separate genera. Moreover, we formulate some hypotheses concerning the life style and origin of the ancestral anemonefish.